[Isosteric triphosphonate analogues of dNTP: synthesis and substrate properties toward various DNA polymerases].

Isosteric triphosphonate derivatives of 2',3'-dideoxy-2',3'-didehydroadenosine and 3'-deoxy-2',3'-didehydrothymidine and their beta,gamma-substituted analogues were synthesized. Their substrate properties toward a number of reverse transcriptases of the human immunodeficiency and bird myeloblastosis viruses, human DNA polymerases alpha and beta, and the Klenow fragment of Escherichia coli DNA polymerase I were studied.

[Nucleotide-dependent degradation of nucleic acids by DNA and RNA polymerases]. / Nukeleotid-zavisimaia degradatsiia nukleinovykh kislot DNK- i RNK-polimerazami.

In this review we summarize the current knowledge about recently discovered reactions of nucleotide-dependent nucleic acid degradation catalyzed by DNA and RNA polymerases. These reactions consist in the excision of the 3'-nucleotide of nascent DNA or RNA chain in the presence of non-complementary r/dNTPs. In the case of DNA polymerases the dinucleoside-5',5"-tetraphosphate as a product of the reaction is formed. In contrast, in the case of RNA polymerases non-complementary r/dNTPs stimulate 3'-->5' exonuclease degradation of RNA transcript. The possible role of these reactions in fidelity of DNA and RNA synthesis, in resistance of HIV reverse transcriptase towards nucleoside inhibitors is discussed.

[Synthesis and substrate properties of modified nucleoside 5'-triphosphates mimicking dATP in the reactions of DNA synthesis catalyzed by DNA polymerases]. / Sintez i substratnye svoistva modifitsirovannykh nukleozid-5'-trifosfatov, modeliruiushchikh dATP v reaktsiiakh sinteza DNK, kataliziruemykh DNK-polimerazami.

Several modified nucleoside 5'-triphosphates were synthesized containing adenine-mimicking pyrimidine derivatives as an aglycone. The study of substrate properties of these compounds towards DNA-synthesizing enzymes showed their ability of being incorporated into the growing DNA chain in place of dATP.

[Substrate properties of dioxolane analogs of 3'-deoxythymidine-5'-triphosphate in DNA synthesis reactions, catalyzed by various DNA polymerases]. / Substratnye svoistva dioksolanovykh analogov 3'-dezoksitimidin-5'-trifosfata v reaktsiiakh sinteza DNK, kataliziruemykh razlichnymi DNK-polimerazami.

The substrate properties of 3'-deoxythymidine 5'-triphosphate analogs prepared on the basis of 2,4-disubstituted 1,3-dioxolanes were investigated in reactions of the DNA synthesis catalyzed by various DNA polymerases. The 4'-triphosphates of (+/-)-cis-4-hydroxymethyl-2-(1-thyminylmethyl)-1,3-dioxolane and the corresponding (+/-)-trans-isomer were shown to be terminating substrates of terminal deoxynucleotidyl transferase. 4'-Triphosphate of (+/-)-cis-4-hydroxymethyl-2-(1-thyminylmethyl)- 1,3-dioxolane terminates the DNA synthesis catalyzed by HIV reverse transcriptase, whereas 2'-triphosphate of (+/-)-cis-2-hydromethyl-4-(1-thyminylmethyl)-1,3-dioxolane is a terminator in the DNA synthesis catalyzed by HIV reverse transcriptase and the Klenow fragment of DNA polymerase I.

[Purine arabinonucleoside-5'-triphosphates with substituents in the 2'-position as DNA polymerase substrates]. / Purinovye arabinonukleozid-5'-trifosfaty s zamestiteliami v 2'-polozhenii kak substraty DNK-polimeraz.

Analogues of araNTPs carrying an azido or aminogroup instead of the 2' hydroxyl exhibited substrate properties towards several mammalian and viral DNA polymerases. At the same time, introduction of a bulky hydrophobic DNP group into the 2' position inactivated the compounds as substrates. HSV-1 and CMV DNA polymerases were an interesting exception: they effectively incorporated the modified nucleotide residues with DNP group into the 3'-termini of the DNA chain. This is a reliable way to distinguish these enzymes from cellular DNA polymerases.

[Modified nucleoside-5'-triphosphates with an additional conjugated through the 2'-3'-fused ring as DNA polymerase substrates]. / Modifitsirovannye nukleozid-5'-trifosfaty s doponitel'nym sopriazhennym po 2'-3'-sviazi tsiklom kak substraty DNK-polimeraz.

5'-Triphosphates of 1-(2',3'-epithio-2',3'-dideoxy-beta-D-lyxofuranosyl) thymine, 1-(2',3'-epithio-2',3'-dideoxy-beta-D-ribofuranosyl) thymine and 2',3'-lyxoanhydrothymidine have been shown to be terminator substrates of human immunodeficiency virus and avian myeloblastosis virus reverse transcriptases as well as DNA polymerase I from E. coli. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase epsilon from human placenta. The KM values of ltTTP, rtTTP and laTTP incorporation into DNA chain agree closely with each other, being 1.5-2.5 times higher than KM for dTTP. Furthermore, Vmax values for modified substrates are only 2-3 times less than Vmax for dTTP. The evidence favours the hypothesis of a great affinity of modified nucleosides with flattened ribose ring of glycone for DNA polymerases active sites.

[5'-S-nucleoside triphosphates. Synthesis and substrate properties in tests with DNA polymerases]. / 5'-S-nukleozidtrifosfaty. Sintez i substratnye svoistva v testakh s DNA-polimerazami.

In the course of our investigations on DNA polymerases a synthesis of new triphosphate analogues, 5'-mercapto-5'-deoxythymidine 5'-S-triphosphate and 5'-mercapto-3',5'-dideoxythymidine 5'-S-triphosphate, was brought about starting from 3'-O-acetyl-thymidine and 3'-deoxythymidine, respectively. The former compound proved to be a weak substrate for E. coli DNA polymerase I and AMV reverse transcriptase and inactive towards DNA polymerases alpha from placenta and beta from rat liver. The second synthesized triphosphate was incorporated into DNA chain by none of the tested enzymes. Probable reasons for so strong a decrease in the substrate properties of the prepared compounds are discussed.

[Acyclic analogs of 2',3'-dideoxy-2',3'-didehydronucleoside-5'-triphosphates--terminators of DNA synthesis, catalyzed by a broad set of DNA polymerases]. / Atsiklicheskie analogi 2',3'-didezoksi-2',3'-didegidronukleozid-5'-trifosfatov--terminatory sinteza DNK, kataliziruemogo shirokim naborom DNK-polimeraz.

O-4'-nor-2', 3'-dideoxy-2', 3'-didehydronucleoside 5'-triphosphates are shown to be effective termination substrates of DNA biosynthesis catalyzed by human placental DNA polymerases alpha and epsilon, rat liver DNA polymerase beta, reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus, and calf thymus terminal deoxynucleotidyl transferase. These compounds do not interact only with the Escherichia coli DNA polymerase I (Klenow fragment). The probable reasons of interaction of acyclo-d4NTP with the DNA synthesizing complexes are discussed.

[Formation of phosphonoester bonds, catalyzed by DNA polymerases]. / Obrazovanie fosfonoéfirnykh sviazei, kataliziruemoe DNK-polimerazami.

3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta, gamma-diphosphate (I) and 2'-deoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma-diphosphate (II) were synthesised. Reverse transcriptases of HIV and avian myeloblastosis virus, rat liver DNA polymerase beta, calf thymus terminal deoxynucleotidyl transferase and E. coli DNA polymerase I KF incorporated both compounds into the growing DNA chain, KF being the least effective. Compound I revealed termination substrate properties, but II was repeatedly incorporated into the DNA chain, for example, by HIV reverse transcriptase - up to 8 residues. Human placenta DNA polymerases alpha and epsilon incorporated neither I nor II into the DNA chain, although DNA synthesis, catalyzed by all the investigated enzymes, was inhibited in the presence of I or II and compound II was a more effective inhibitor then I. The DNA fragments containing alpha-phosphonomethyl groups were hydrolyzed by 3'----5' exonuclease of DNA polymerase I and not hydrolyzed by ExoIII from E. coli.

[Inhibition of various stages of DNA replication in sea urchin embryos]. / Ingibirovanie otdel'nykh stadii protsessa replikatsii DNK émbrionov morskikh ezhei.

The embryos of the sea urchin Strongylocentrotus intermedius possess the ability to incorporate into their DNAs 2'-deoxynucleosides together with all their bases, i.e., adenine, guanine, cytosine and thymine. This incorporation is inhibited by 3'-amino-2',3'-dideoxynucleosides with the same bases. 5'-Amino-5'-deoxynucleosides and 5'-amino-2',5'-dideoxynucleosides moderately inhibit the incorporation of [3H]2'-deoxynucleosides into the DNAs by competing with the latter, presumably at the phosphorylation stage. The most potent inhibiting effect is exerted by 2'-amino-2'-deoxynucleosides and 2'-asido-2'-deoxynucleosides; the mechanism of this inhibition is still obscure, however.

[The peptidyltransferase center of ribosomes--what is it?]. / Peptidiltransferaznyi tsentr ribosom--chto zhe éto takoe?

The recent data on the interaction of model substrates and substrate-like analogs with acceptor and donor sites of 70S and 80S ribosomes are considered in terms of peptidyl transferase center models suggested earlier.

[Interaction of substrates and substrate-like inhibitors with the peptidyltransferase center from Escherichia coli ribosomes]. / Vzaimodeistvie substratov i substratpodobnykh ingibitorov s peptidiltransferaznym tsentrom ribosom Escherichia coli.

The binding isotherms of CACCA(3'NHPhe----Ac) and CACCA(3'NHPhe) to E. coli ribosomes and 50S subunits were measured. A theoretical model of adsorption for the case of cooperative interaction between two ligands adsorbed on a ribosome was designated. The analysis of the experimental binding isoterms leads to the following conclusions. A ribosome (or subunit) binds one CACCA (3'NHPhe----Ac) molecule to donor site of the peptidyl transferase center, but two CACCA (3'NHPhe) molecules to both donor and acceptor sites. The binding of CACCA (3'NHPhe) to ribosomes (or subunits) is a cooperative process, characterized by the cooperativity coefficient tau = 40 +/- 5 or more. When model substrates CACCA-Phe, CACCA-Leu and CACCA-Val were taken instead of CACCA (3'NHPhe) in the incubation mixture with ribosomes, dipeptides were obtained even in the case, when ratio [model substrate]: [ribosome] (in moles) was much lower than 1. Puromycin binding to acceptor site with constant (1-2) X 10(4) M-1 also stimulates CACCA(3'NHPhe----Ac) adsorption to the donor site of ribosomes with cooperativity coefficient being equal to 1.5-2.5. It is also shown that cytidine 5'-phosphate binding to the donor site increases kappa cat of the reaction of minimal donors with CACCA-Phe by 1.5 orders of magnitude but has no effect on Km of this reaction. These facts point out that cytidine 5'-phosphate being adsorbed on the corresponding area of the donor site leads to the conversion of low-productive complex [ribosome + minimal donor substrate + acceptor substrate] into high-productive complex [ribosome + minimal donor substrate + acceptor substrate + cytidine 5'-phosphate].

[Action of sulfochlorantine on cell-free systems of Escherichia coli]. / O deistvii "Sul'fokhlorantina" na beskletochnye sistemy Escherichia coli.

Sulfochlorantine containing active chlorine was shown to produce a general toxic action inactivating a number of Escherichia coli enzyme systems involved in protein biosynthesis. DNA synthesis catalysed by calf thymus DNA polymerase alpha was repressed by 50% and the synthesis of aminoacyl-tRNA catalysed by aminoacyl-tRNA synthetases was repressed by 70% in the cell-free systems at the lethal concentration of the bactericide (0.01% and higher). Sulfochlorantine produced the strongest inhibiting action on the ribosomal step of protein biosynthesis, inhibiting the poly(U)-directed polyphenylalanine formation by 95% at the sublethal concentration of the bactericide (0.005%).

[Effect of sulfochloranthine on protein and nucleic acid biosynthesis in Escherichia coli]. / Vliianie sul'fokhlorantina na biosintez belka i nukleinovykh kislot Escherichia coli.

Sulfochloranthine was shown to be bacteriostatic for Escherichia coli B cells grown in a chemically defined medium at a concentration of 0.002%, sublethal at a concentration of 0.005%, and lethal at 0.01% (0.000312, 0.00078 and 0.00156% of active chlorine, respectively). Protein synthesis by E. coli B cells was noticeably inhibited when the concentration of the preparation was 0.002%, and stopped completely at a 0.01% concentration of the preparation. Biosynthesis of nucleic acids, in particular DNA, was inhibited to a lesser extent. The bacteriostatic concentration of the preparation had virtually no effect on DNA biosynthesis, but inhibited RNA biosynthesis by 50%. Sulfochloranthine used at sublethal doses inhibited synthesis of both DNA and RNA by 75%; DNA and RNA biosynthesis ceased at the lethal concentration of the preparation.

[Donor site of E. coli ribosomal peptidyltransferase]. / Issledovanie donornogo uchastka peptidiltransferazy ribosom E. coli.

The mechanism of 5'-cytidilic acid stimulation of the reaction between 2'(3')-O-formylmethionine ester of 5'-adenylic acid and phenylalanyl-tRNA catalyzed by E. coli ribosomes has been studied. It has been shown that cytidilic acid binds to the donor site of the peptidyltransferase in the area which is usually occupied by the second nucleotide residue of the peptidyl-tRNA 3'-end. After the binding cytidilic acid stimulates effectively the donor activity of formylmethionine ester of adenylic acid. A number of compounds have been tested as possible stimulants. Both the chemical nature of stimulant and its conformation are important for the stimulating action. A hypothetic scheme is suggested explaining possible causative factors of peptidyl-tRNA translocation from the acceptor site to the donor site after peptide bond formation.

[Interaction of dansyl-dipeptidyl-tRNA with E. coli ribosomes]. / Osobennosti vzaimodeistviia dansil-dipeptidil-tRNK s ribosomal E. coli

Dansyl-dipeptidyl-tRNA is found to bind with ribosomes in the presence of polyU much more efficiently than AcPhe-tRNA. 30-80% OF introduced danysl-dipeptidyl-tRNA is in a complex with ribosomes and reacts with puromycin in the presence of 5 mM Mg2+. A significant binding of dansyl-dipeptidyl-tRNA both for acceptor and donor ribosome regions is observed in the presence of 10 mMg2+, unlike AcPhe-tRNA, which is capable of the binding only at 20 mM Mg2+. A peptide transfer from dansyl-dipeptidyl-tRNA on puromycin takes place at 10 mM Mg2+ in the absence of template, while AcPhe-tRNA efficiently interacts with puromycin only at 20 mM Mg2+. The data obtained suggest that the increase of hydrophobity of a peptide residue in peptidyl-tRNA increases its binding in peptidyl transferase of ribosomes.